Glutamate transporters remove glutamate from synapses to maintain an efficient synaptic communication between neurons and to prevent extracellular glutamate concentrations from reaching neuro-toxic levels. The long-term goal of this project is to understand the molecular mechanism of glutamate transporter. It is important to understand the molecular mechanism by which glutamate transporters achieved glutamate uptake, because this will lead to a better understanding of synaptic communication in the brain, as well as, lead to treatments to prevent neuronal death due to glutamate toxicity during, for example, ischemia. In addition, this study will provide essential structural and dynamical information about the glutamate transporters, an area of research in which there are only a few numbers of groups working. Our current knowledge of the structure and function of glutamate transporters is very limited. No structural model has yet been suggested that can explain how glutamate transporters accomplish glutamate uptake. We propose to use fluorescence and biochemical methods to elucidate the structure and function of glutamate transporters, using fluorescent and non-fluorescent cysteine reactive probes. These techniques allow us to obtain structural as well as dynamical information about the molecular mechanism of glutamate transporters. Our objective is to construct a structural model of glutamate transporters and to elucidate how changes in this structure lead to glutamate transport. The aims of the proposed project are: 1) To identify the domains of the glutamate transporters that undergo conformational changes during the transport cycle; and 2) To identify residues that undergo alternating access during glutamate transport.